Gene identification and allele-specific marker development for two allelic low phytic acid mutations in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is an important anti-nutritional component in cereal and legume grains. PA forms of phosphorus (P) and its salts with micronutrient cations, such as iron and zinc, are indigestible in humans and non-ruminant animals, and hence could affect food/feed nutritional value and cause P pollution of ground water from animal waste. We previously developed a set of low phytic acid (LPA) rice mutants with the aim to increase their nutritional quality. Among them, one line, i.e., Os-lpa-XQZ-1 (hereafter lpa 1-2), was identified to have a mutation allelic to the KBNT lpa 1-1 mutation (hereafter lpa 1-1), which was already delimited to a 47-kb region on chromosome 2. In this study, we searched the candidate gene for these two allelic LPA mutations using T-DNA insertion mutants, mutation detection by CEL I facilitated mismatch cleavage, and gene sequencing. The TIGR locus LOC_Os02g57400 was revealed as the candidate gene hosting these two mutations. Sequence analysis showed that the lpa 1-1 is a single base pair substitution mutation, while lpa 1-2 involves a 1,475-bp fragment deletion. A CAPS marker (LPA1_CAPS) was developed for distinguishing the lpa 1-1 allele from lpa 1-2 and WT alleles, and InDel marker (LPA1_InDel) was developed for differentiating the lpa 1-2 allele from lpa 1-1 and WT ones. Analysis of two populations derived from the two mutants with wild-type varieties confirmed the complete co-segregation of these two markers and LPA phenotype. The LOC_Os02g57400 is predicted to encode, through alternative splicing, four possible proteins that are homologous to the 2-phosphoglycerate kinase reported in hyperthermophilic and thermophilic bacteria. The identification of the LPA gene and development of allele-specific markers are of importance not only for breeding LPA varieties, but also for advancing genetics and genomics of phytic acid biosynthesis in rice and other plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.)

In our previous cDNA microarray analysis, we identified 53 mature anther-specific genes, whose function was unknown, in rice. We reanalyzed these genes from the viewpoint of the specific amino acid motif. Out of 53 genes, three genes, Os-26, Os-32, and Os-169 (renamed as OsSCP1, OsSCP2, and OsSCP3), encoded cysteine-rich motif (Cys-X₃-Cys-X₁₃-Cys-X₃-Cys), indicating that they were novel small cysteine-rich proteins. From the search of specific elements in promoter regions, several pollen-specific elements were found. In order to determine whether three promoters were functional in pollen or not, the gene constructs with promoter regions fused to the β-glucuronidase gene were transformed into tobacco. Histochemical analysis showed that these promoters were active in the mature pollen grains and pollen tubes. Furthermore, OsSCP1 and OsSCP3 formed a multigene family tandemly in the rice genome. From the results, OsSCPs might have important roles in mature pollen development and pollen tube growth.     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

Molecular cloning and nucleotide sequence cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

QTLs and epistasis underlying activity of acid phosphatase under phosphorus sufficient and deficient condition in rice (Oryza sativa L.)

Eighty-four selected lines from a recombinant inbred (RI) line population of 284 lines derived from a cross between the indica varieties IR20 and IR55178-3B-9-3 were used in a hydroponic culture experiment with sufficient P supply (10 mg P L^–1) and P-deficient stress (0.05 mg P L^–1). After 2 weeks, the activity of acid phosphatase (AAP) in roots of each parent and each line from both normal culture and P-deficient stress was determined. QTLs for AAP, P-deficiency stress induced AAP (Psi-AAP) and relative AAP (RAAP) were detected using 178 molecular markers mapped on all 12 chromosomes based on single marker analysis and interval mapping. One QTL for AAP and three QTLs for Psi-AAP were detected on chromosome 1, 6 and 12, respectively. Two QTLs for RAAP were identical with these for Psi-AAP on chromosome 6 and 12. The results in this case indicated that the genetic system for Psi-AAP was different with that for AAP under normal culture. The AAP was mainly influenced by interaction among muti-factors, while Psi-AAP was controlled by a Psi genetic system.     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice ( Oryza sativa L.)

By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three cDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine decarboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (namedREF1 A), and a novel gene whose function is unknown (namedSRG1). The full-length cDNA of SAMDC gene (namedSAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and buman. Northern hybridization revealed that expression of SAMDCl and REFlA was induced, while SRGl was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDCl and SRGl were present as a single copy gene in rice genome, whereas riceREF1 A gene was organized as a gene family. TheREF1 A,SAMDC1, andSRG1 genes were located on chromosome 3,4, and 6 respectively by RFLP mapping approach using ZYQ8/JX17 DH population and RFLP linkage maps. Project supported by the National “863” High-Technology Program.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine

Among the mutant lines of rice that have been selected for morphological characters, one line, NM67, was found to have a low content of glutelin and a higher content of prolamine in its seed protein than other Japanese cultivars. This mutant is a semi-dwarf and partially sterile line, and its leaves turn yellow before heading. Genetic analysis after backcross to the original cultivar, Nihonmasari, revealed the following: (1) the character of low glutelin content was always accompanied by the character of high prolamine content; (2) the low glutelin (and high prolamine) character seemed to be manifested by a single dominant gene; and (3) semi-dwarfness, low fertility and early yellowing leaf of the mutant, which might also be pleiotropy, were controlled by a single recessive gene independent of the gene for protein content. The protein character of NM67 was genetically separated from semi-dwarfness and low fertility, and a new line having low glutelin content and high prolamine content with normal morphological characters comparable to those of the original cultivar was obtained from the progenies of the cross. The possible use of this line as a low protein rice cultivar is discussed.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.)

A single dominant gene Gc controls the trait of high chlorophyll (Chl) content in rice (cultivar (cv.) Zhenshan 97B). The contents of Chl b and total Chl increased 100% and 25%, respectively, when Gc was introduced. In addition, photosynthetic rate, biomass and grain yield also increased by 20%, 17% and 16%, respectively. Three simple sequence repeats (SSR) markers (rm462, rm6340 and rm6464) that are linked to Gc were identified by amplification of DNA samples from near-isogenic lines using two hundred pairs of primers. The genetic distances on the short arm of rice chromosome 1 between Gc and rm6464, rm6340 and rm462 were 0, 0.588 and 1.18 cM, respectively.     

Inheritance and mapping of embryo sac abortion in hybrid between Indica and Japonica rice (Oryza sativa L.)

A doubled haploid (DH) population derived from anther culture of F₁’s between an Indica var. Zhai-Ye-Qing 8 and a Japonica var. Jing-Xi17 as well as two backcross populations derived from this DH population were used to investigate inheritance of the embryo sac abortion at early megagametogenesis occurring in Indica/Japonica rice crosses. Two major loci, dominant and complementaryesa-1 andesa-2, located on chromosomes 6 and 12 respectively, were detected. Genetic analysis indicated that embryo sac fertility is mainly regulated by the gametophytic genotype at these two loci.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Diallel analysis of heading date in rice (Oryza sativa L.)

Summary The genetics of heading date was investigated in an 8×8 diallel set of crosses involving diverse rice cultivars. Wr, Vr graph analysis revealed the presence of a complementary type of non-allelic interaction which apparently affected the position and slope of the regression line such as if there were overdominance. Omission of two interacting parents resulted in a 6×6 subset of diallel crosses from which, as observed in the Wr, Vr graph, the non-allelic interaction had disappeared and the regression line exhibited partial dominance. Estimates of the genetic components of variation were in close conformity with the results obtained from the Wr, Vr graph: the average degree of dominance, as measured by (H₁/D)^1/2, was in overdominance range in the interacting 8×8 set of diallel crosses whereas it was reduced to partial dominance in the non-interacting 6×6 set of crosses. Further analysis by a standardized deviations graph indicated that earliness was controlled, on the average, by an excess of dominant alleles.     

Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.)

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring -phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.     

Response of excised embryos of rice (Oryza sativa L.) to X-rays

Summary The response of rice (Oryza sativa L.) embryos to X-rays (M₁ to M₃) was studied. By means of irradiating excised embryos, both chlorophyll and macromutation were successfully induced in three genotypes of rice. However, differential responses in terms of mutation frequency, mutation spectrum and optimal levels of X-rays required for induction of mutation (chlorophyll as well as morphological) were found to exist between cultivars. In Satika and Ashkhata, LD₅₀ values and maximum induced seed sterility are concomitant to optimum level of radiation required for triggering chlorophyll mutation. However, optimum dose for induction of macromutation in Satika and Kerangserang is independent of either LD₅₀ and/or induced seed sterility.Chances of obtaining both dominant and locus specific recessive mutations in the immediate X-ray treated generation (M₁) are large. This indicates the very high degree of effectiveness of the excised embryo irradiation technique with rice.